Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities.
Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.
Palosaari
P M
PM
Department of Medical Biochemistry, University of Oulu, Finland.
Hiltunen
J K
JK
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase; D004746:Enoyl-CoA Hydratase / Q000302:isolation & purification / Q000378:metabolism*; D006836:Hydro-Lyases / Q000378:metabolism*; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D063994:Peroxisomal Bifunctional Enzyme; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase